The present disclosure relates to the field of cell, tissue and organ preservation at low temperatures (e.g., cryopreservation). More specifically, the present disclosure relates to methods for treatment of cellular materials with glycolipids, such as, for example, antifreeze glycolipids (AFGLs) that have been isolated from freeze-tolerant/freeze-avoiding organisms and/or plants, and provides a cell population with enhanced cell survival post-preservation—even under suboptimal cooling conditions, such as rapid cooling.
Conventional approaches to cryopreservation generally include addition of 10% DMSO to cells in suspension in cryovials and slow rates of cooling, with or without induced nucleation, and storage at −80° C. or below −135° C. As long as viable cells are present upon thawing, cell yield is often a secondary consideration in conventional approaches. However, there are cell types and tissues that are difficult to preserve and situations where cell yield is critical such as for cell therapy applications. Alternative protocols and solutions that improve cell viability and yield (even under suboptimal cooling conditions) and allow for the preservation of cell types that are traditionally hard to preserve are needed.
Nature has developed a wide variety of alternative strategies for allowing organisms/plants to tolerate/survive extreme temperatures. The study of how organisms/plants survive extreme temperatures has revealed that they produce various antifreeze compounds, which help them either avoid freezing or tolerate freezing until warmer temperatures are available. For example, the discovery of antifreeze proteins in fish and insects has provided an avenue to explore alternative preservation strategies.
In this regard, it has been found that the presence of antifreeze proteins lowers the freezing point of the solution and also changes the shape and formation of ice. For example, anti-freeze peptides (AFPs) can adsorb to the surface of ice crystals, blocking the addition of water molecules to growth sites, which decreases the temperature at which the crystal grows (called the hysteretic freezing point) by as much as 13° C. AFPs can also bind to embryonic ice crystals, thereby inhibiting ice nucleation and permitting extensive supercooling well below the freezing point. They are also thought to be able to modify ice structure, inhibit recrystallization and modify the fluid properties of solutions, thereby extending survival of organisms in subzero environments. In this way, potentially less cryoprotectant may be used to preserve cells reducing potential cytotoxicity.
While anti-freeze peptides (AFPs) are known and have been studied extensively, various anti-freeze glycolipids (AFGLs) have also been found in several organisms/plants, including insects (both freeze tolerant insects and freeze avoiding insects), and freeze tolerant plants. However, prior to the methodology of the present disclosure, the use of such larger molecules in the context of preserving mammalian cells had been somewhat limited (in part because mammalian cell membranes are believed to be impermeable to larger molecules (Castro, A. G., Lapinski, J., Tunnacliffe, A. Nature Biotechnology 18:473, 2000) and it was previously believed that for molecules to be effective at enhancing survival, such molecules need to be present both on the inside and the outside of the cell membrane). The isolation of such AFGLs provides an opportunity to develop/enhance preservation methods for a variety of cell and tissue types.